Genome characteristics and the ODV proteome of a second distinct alphabaculovirus from Spodoptera litura

Background Spodoptera litura is a harmful pest that feeds on more than 80 species of plants, and can be infected and killed by Spodoptera litura nucleopolyhedrovirus (SpltNPV). SpltNPV-C3 is a type C SpltNPV clone, that was observed and collected in Japan. Compared with type A or type B SpltNPVs, SpltNPV-C3 can cause the rapid mortality of S. litura larvae. Methods In this study, occlusion bodies (OBs) and occlusion-derived viruses (ODVs) of SpltNPV-C3 were purified, and OBs were observed by scanning electron microscopy (SEM). ODVs were observed under a transmission electron microscope (TEM). Results Both OBs and ODVs exhibit morphological characteristics typical of nucleopolyhedroviruses (NPVs).The genome of SpltNPV-C3 was sequenced and analyzed; the total length was 148,634 bp (GenBank accession 780,426,which was submitted as SpltNPV-II), with a G + C content of 45%. A total of 149 predicted ORFs were found. A phylogenetic tree of 90 baculoviruses was constructed based on core baculovirus genes. LC‒MS/MS was used to analyze the proteins of SpltNPV-C3; 34 proteins were found in the purified ODVs, 15 of which were core proteins. The structure of the complexes formed by per os infectivity factors 1, 2, 3 and 4 (PIF-1, PIF-2, PIF-3 and PIF-4) was predicted with the help of the AlphaFold multimer tool and predicted conserved sequences in PIF-3. SpltNPV-C3 is a valuable species because of its virulence, and the analysis of its genome and proteins in this research will be beneficial for pest control efforts. Supplementary Information The online version contains supplementary material available at 10.1186/s12864-024-09989-3.

Genome characteristics and the ODV proteome of a second distinct alphabaculovirus from Spodoptera litura Background Baculoviridae is the largest viral family; it consists of rod-shaped viruses specific to arthropods.It is a type of enveloped virus with a circular double-stranded DNA genome ranging in size from 80 to 180 kb [1] and containing 100 to 180 open reading frames [2].Baculoviruses were among the first species of insect viruses discovered, and more than 600 insect species across 7 orders, such as Lepidoptera, Hymenoptera, and Diptera, have been reported to be infected by baculoviruses.To date, 91 complete genomes have been recorded in National Center for Biotechnology Information (NCBI) database; these include four genera: Alphabaculovirus (61), Betabaculovirus (26), Gammabaculovirus (3), and Deltabaculovirus (1) [3].
Over the course of coevolution, baculoviruses have evolved two types of virions, budded viruses (BVs) and occlusion-derived viruses (ODVs), during their life cycle to enhance the ability to infect the host.One or more ODVs are released from occlusion bodies (OBs) in an alkaline environment such as the gut of an insect; ODVs are thus released, and subsequently infect midgut epithelial cells [4].After infecting midgut epithelial cells, BVs are packaged and released to disseminate systemic infection from cell to cell.In the next several days, the larvae dissolve from the inside and release the OBs.Because of this trait, since the 1940s, baculoviruses have been studied as biopesticides in crop fields.Although baculoviruses cannot kill insect larvae quickly, baculoviruses are still targeted, environmentally friendly and low-cost biopesticide.
Spodoptera litura, which belongs to the family Noctuidae and is called the tobacco cutworm or cotton leafworm, is a nocturnal moth found across Asia, Oceania, and the Indian subcontinent.Larvae eat indiscriminately and voraciously, and thus pose a threat to cash crops.Using chemical insecticides is not friendly to the environment, and insecticidal lamps are mainly aimed at imagines.In contrast, using baculoviruses as biopesticides is a good choice.Spodoptera litura nucleopolyhedrovirus (SpltNPV) is widely found in Central Asia, including China, Japan and Pakistan [5], and has been successfully applied as a commercial biopesticide against defoliating insects in China.An analysis of samples collected by Kamiya in Japan identified three NPV types, as type A, type B, and type C. A clone from type C SpltNPV called SpltNPV-C3 could cause more rapid mortality of S. litura larvae than type A or type B SpltNPV [6].
It is important to analyze the genome and predict the structure of proteins information for determining the lethality of baculovirus and identifying the host domain.In this study, the genome of SpltNPV-C3 was analyzed, OBs and ODVs were purified and observed via electron microscopy, proteins were separated via LC-MS/MS, and simulated structures were connected and associated with oral infection.

Virus preparation and purification
SpltNPV-C3was a gift from Jiang Zhu (Soochow University), and was originally obtained from Katsumi Kamiya (Gifu Prefectural Institute for Bio-Industrial Technology, Minokamo, Japan).
S. litura larval corpses were collected, ground in a mortar with PBS and filtered through cheesecloth.The collected filtrate was centrifuged at 500 rpm (30 × g) for 10 min (Hitachi CF15RX II), followed by pelleting with centrifugation via 8000 rpm (7100 × g) for 30 min, washing the sediment at 3000 rpm (1000 × g) for 20 min with PBS three times, collecting the sediment at 8000 rpm for 30 min, and storing it at 4 ℃.
ODVs were collected from liquefied larvae.Freshly purified OBs of SpltNPV-C3 suspended in ddH 2 O were incubated with an equal volume of lysis buffer (0.3 M Na 2 CO 3 , 0.5 M NaCl, and 0.03 M EDTA, pH 9.5) at 37 °C for 10 min.The pH was adjusted to 7.5 with 0.1 M HCl.The viral OBs were purified by differential centrifugation.The released ODVs were purified via a 30-60% discontinuous sucrose gradient by centrifugation at 100,000 × g (Hitachi CS150GX II) for 90 min at 4 ℃.The collected ODVs were washed in 0.1× TE (10 mM Tris-HCl and 1 mM EDTA, pH 7.5) by centrifugation at 40,000 × g at 4 ℃ for 1 h.The sediment was resuspended in 0.1× TE [7].

DNA sequencing and analysis
A random genomic library of SpltNPV-C3 was constructed according to the partial filling-in method (Chen et al., 2009) [9].ORFs were defined using ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html).DNA and protein comparisons were performed using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi).Protein homology and translated ORFs were identified by the HHpred webserver [10,11].Multiple alignments and percentage identities were obtained using ClustalW.Putative ORFs were screened as described previously [12].Phylogenetic analysis of SpltNPV-C3 was conducted through a phylogenetic tree based on the amino acid sequences of the core genes of Baculoviridae available in the ICTV (https://talk.ictvonline.org/ictv-reports/ictv_online_report/dsdna-viruses/w/baculoviridae) using the maximum likelihood method and tested by the bootstrap method in MEGA X.Late expression factor 8 (lef-8), late expression factor 9 (lef-9) and polyhedrin (polh) were seriated and used to calculate the genetic distances via MEGA (Kimura two-parameter model) [13].

Protein separation and in-gel digestion
The proteins of the SpltNPV-C3 OBs were separated via SDS-PAGE using an 8-15% gradient gel.The protein bands were collected into a 1.5 mL centrifuge tube for LC-MS/MS analysis (Thermo Fisher Scientific, MA, USA).LC-MS/MS analysis and protein identification were performed by Shanghai Omicsolution Co.The raw files of the MS spectra were searched against the putative protein database SpltNPV-C3 (NC_011616).

Protein structure simulation
The amino acid sequences used were found in the NCBI genome database Complete genomes: Baculoviridae (nih.gov).The 3D structure was simulated by the AlphaFold Multimer tool.Conserved sequences were estimated by The ConSurf Server (tau.ac.il).

Electron microscopy observation
Polyhedrin envelops ODVs to protect them from extraneous harmful environmental risks.Previous research has shown the physical form of baculoviruses.With the help of SEM, our results showed that the OBs of Splt-NPV-C3 are packaged with spherical polyhedra that have an uneven surface and are approximately 1.5 μm in diameter, in accordance with the standard mode of Alphabaculovirus.The structure of the virus is shown in Figs. 1  and 2.

Sequence and genome characteristics of SpltNPV-C3
The whole genome of SpltNPV-C3 is 148,634 bp (Gen-Bank accession 780,426, which was submitted as Splt-NPV-II) with a G + C content of 45%.The SpltNPV-C3 genome is 9 kb longer than the SpltNPV-G2 genome (139,342 bp) [16] and 4 kb longer than that of the first sequenced baculovirus, AcMNPV (Autographa California multiple nucleopolyhedroviruses) (133,894 bp).According to the general criteria for discriminating ORFs [17,18], 149 ORFs were found.The number of predicted ORFs and the length of the genome are similar to those of AgseNPV (Agrotis segetum nucleopolyhedrovirus) (151).Among these predicted ORFs, 24 contain early promoter motifs (a CAG/TT motif downstream of the TATA box and within 180 bp upstream of the start codon ATG), 55 contain late promoter motifs (an (A/T/G) TAAG motif downstream within 180 bp upstream of the start codon ATG), and 18 contain early and late promoter motifs, implying that these genes can be transcribed in the early and late stages of viral infection; 51 have no typical motifs for distinguishing early or late characteristics and are difficult to classify.The reading frames and homologous repeat regions are shown in Table 1.

Comparison of SpltNPV-C3 predicted ORFs to those of other baculoviruses
By comparing the gene organization and homology between SpltNPV-C3 and other baculovirus genomes, additional information can be obtained to determine the diversity of baculoviruses and gene evolution.Splt-NPV-C3 has 149 predicted open reading frames (ORFs), including 38 core baculovirus genes [19].In contrast with other baculoviruses, SpltNPV-C3 shares 101 ORFs with AcMNPV, and it is estimated that these ORFs constitute approximately 67.8% of the total.Ninety-eight ORFs are homologous to SpltNPV-G2 ORFs, lower than the 103 of SeMNPV and 141 of SperNPV.SpltNPV-C3 and Splt-NPV-G2 were found in the same host, but the homology between them was lower than that between SeMNPV and SperNPV.In this study, SpltNPV-C3 ORF26, ORF27, ORF28, ORF34, ORF72, ORF89, ORF90, and ORF108 were found only in SpltNPV-C3, not in SperNPV, and ORF26, ORF27, ORF34, ORF108 had no homologs in other baculoviruses (Table 1).Protein homology analysis via BLAST revealed that these four unique ORFs had no recognizable promoter.The specific functions of these proteins may be revealed in future studies.
The whole genome of SpltNPV-C3 was compared with that of SpltNPV-G2 (NC_003102), and the percentage identity was 76.42%, which was lower than that of AcMNPV (80.20%),SeMNPV (NC_002169) (84.63%) and SperNPV (NC_055502) (96.10%).These viruses belong to the Alphabaculovirus genus.and their names originate from their hosts.The SpltNPV-C3 genome is most closely related to the SperNPV genome.The host of SpltNPV is Spodoptera litura, which is distributed across Asia and Oceania.Spodoptera eridania is the host of SperNPV found across North America, and it will be interesting to thoroughly investigate the discrepancy between Splt-NPV and SperNPV caused by regional disparity.Splt-NPV is similar to other viruses.Research has shown that BmNPV (Bombyx mori nucleopolyhedrovirus) has 93% homology with AcMNPV but lacks homologs of Ac3, Ac7 (orf603), Ac48, Ac49, Ac70, Ac86, and Ac134.Ac7 (orf603) is related to lethal genes and cannot be found in SpltNPV-C3 either; this is a universal phenomenon in the baculovirus family likely because these viruses have Fig. 2 Transmission electron micrographs of SpltNPV-C3 ODVs.Magnification is indicated at the bottom of the image.A: 60 000×, B: 25 000×.The images were created using electron micrographs, and the whole appearance of SpltNPV-C3 OBs is clearly shown.Most OBs consist of polyhedrin, which is important for protecting baculoviruses from harsh environments until the next host is found.Baculoviruses can be used as delivery vectors since their genome can contain a long exogenous gene, and viruses produced from larvae can survive against complement attack [14], whereas those packaged by cells cannot survive [15].There is an obvious difference between these two production methods; viruses in larvae experience the whole cycle of baculovirus infection and produce OBs when they exit the larval body   the same ancestor and lost these genes during evolution.These deletions may cause differences in hosts [20].
For viruses, a stronger lethality reduces the chance of survival.Causing rapid mortality of S. litura larvae is a disadvantage for generating a descendant virus.Hosts can evolve defense mechanisms to protect themselves from viruses; it is thus beneficial for viral genes to mutate more quickly.Producing more mutants leads to more chances to overcome the host's defense.There is a set of common genes that cannot be changed; these genes are called core genes, and seem to be crucial factors for some main biological functions.Core genes control the fundamental components of baculoviruses.Other genes that transform are present in different forms in different baculoviruses probably contain confer the secret of evolutionarily advantageous functions.

Phylogenetic analysis of SpltNPV-C3
Genome analysis revealed 38 conserved genes in baculoviruses, all of which can be found in SpltNPV-C3.The phylogenetic analysis was based on the 38 core-gene amino acid sequences from SpltNPV-C3 and the other 89 baculoviruses that were collected and listed in ICTV (https://ictv.global/report/chapter/baculoviridae/baculoviridae) using the maximum likelihood (ML) method with 1000 bootstrap replicates.With the phylogenetic Fig. 3 Phylogenetic tree of 90 baculoviruses with complete sequences.A phylogenetic tree was generated using MEGA X software via the maximum likelihood method and the JTT matrix-based model.The results were visualized using iTOL [ tree, SpltNPV-C3s were classified into an Alphabaculovirus clade, with a shorter genetic distance between SperNPV and SeMNPV.A phylogenetic tree of 90 baculoviruses with complete sequences is shown in Fig. 3.

Virus species demarcation criteria
Traditional naming rules give precedence to the host origin.Thus, unreliable identification sometimes occurs.For example, the same virus extracted from different hosts is given a different name.With the help of molecular biology, a phylogenetic species criterion for Lepidopteraspecific baculoviruses that uses the genetic distances of the partial lef-8, lef-9, and polh genes has been established by an increasing number of researchers.Generally, baculoviruses are considered to belong to the same species when the distance lower than 0.015 according to the Kimura 2-parameter model [13].The distances among AcMNPV, SeMNPV, SperNPV, SpltNPV-G2, and Splt-NPV-C3 were determined.The results showed that the distance between SpltNPV-C3 and SperNPV was 0.0156, which indicated that they were closely related but still two different species.Different data were obtained when the lef-8, lef-9, and polh genes were separated and when calculating the genetic distance alone.The distance of was 0.0163 for lef-8, and 0.0222 for lef-9, the sequence of polh was identical, i.e., a distance of 0. SpltNPV-C3 and SpltNPV-G2 do not have the greatest similarity, which implies that the classifications that exist at present cannot demonstrate true relationships among baculoviruses.Interestingly, that SpltNPV-C3 and SpltNPV-G2 can infect the same insects, but their genes are not very close.In terms of the core genes, SpltNPV-C3 is very close to SperNPV, but they can infect different insects.Therefore, some important genes can influence the virus's choice of host.Table 2 shows the distances of the nucleotide sequences.

Protein analysis via LC-MS/MS and structure prediction
Table 3 shows the identified ODV proteins of SpltNPV-C3.According to previous research, the multiprotein complex of per os infectivity factors (PIFs) is indispensable for baculovirus infection of insect midgut cells.odv-e56, PIF-1, PIF-2, PIF-3, odv-ec43, p48, p40, 38 K, odv-e25, vp39, vlf-1, desmop, vp1054, odv-e27, and p49 are core genes and were detected by LC-MS/MS, a tool for routine protein identification.Purified ODVs were separated via SDS-PAGE, and the resulting peptides were analyzed via LC-MS/MS.Thirty-four proteins were identified, 15 of which were core baculovirus genes [22].Interestingly, PIF-1, PIF-2, PIF-3, and PIF-4 can form a complex, but only PIF-1, PIF-2, and PIF-3 were detected in our LC-MS/MS results, where the disposition of PIF-4  Core gene; Required for BV production is unknown.The PIF-4 protein was not detected via LC-MS/MS, possibly due to its low expression level.odv-e56 (PIF-5) is also an important protein for baculoviral oral infectivity (Li, et al., 2022).This paper reveals the essential role of intramolecular interactions in baculoviral oral infectivity.https://doi.org/10.1128/jvi.00806 − 22).Other PIF proteins were not detected by LC-MS/MS, and these proteins may be the cause of larval infection during the OB period.
This result is interesting because it shows that PIF-1 and PIF-2 are related to recognizing the host and that some parts of PIF-3 can help the virus infect midgut cells.We simulated the 3D structure of SpltNPV-C3 PIF-1, PIF-2, PIF-3, and PIF-4 and calculated the conserved amino acids by multiple sequence alignment on The ConSurf Server (tau.ac.il).After contrasting AcMNPV, BmNPV, SperNPV, SeMNPV, and SpltNPV-G2, we discovered a conserved sequence on the tail of PIF-3.The structure predicted by AlphaFold 2 is shown in Fig. 4.
After the PIF-3 model was constructed, the Alpha-Fold Multimer tool was used to predict the model of the PIF-1, PIF-2, PIF-3, and PIF-4 complex.The red region is a conserved sequence located in the middle of the PIF complex.These amino acids are preserved throughout evolution, and thus this region may correlate well with  the infecting larval midgut.The structure of the PIF-1, PIF-2, PIF-3, and PIF-4 complexes were simulated by the AlphaFold Multimer tool, as shown in Fig. 5.

Conclusion
The morphological characteristics of purified OBs and ODVs of SpltNPV-C3 were examined morphological characteristics under EM.The OBs of SpltNPV-C3 are approximately 1.5 μm in diameter, and the ODV is approximately 300 nm in length and 40 nm in width.The whole genome of SpltNPV-C3 is 148,634 bp (GenBank accession 780,426), with a G + C content of 45%, and 149 ORFs were found.Using the ML method, a phylogenetic tree of 90 baculoviruses was constructed, and SpltNPV-C3 was found to belong to the Alphabaculovirus group and was most closely related to SperNPV according to our tree.Thirty-four proteins were found in the purified ODVs, 15 of which were core genes in the family Baculoviridae.The complex of PIF-1, PIF-2, PIF-3, and PIF-4 was simulated by the AlphaFold Multimer tool, and a conserved sequence of PIF-3 was found in the middle of the PIF complex.This research is helpful for studying baculovirus infection and the origin of the baculovirus family.

Note:
Putative SpltNPV-C3 predicted ORFs are listed in Column 1, and the gene homologs are listed in Column 2. Column 3 indicates the ORF location and transcriptional direction in the SpltNPV-C3 genome.Column 4 indicates the number of amino acids.Column 5 indicates the presence of early (E) and/or late (L) promoters located upstream of the start codon of each ORF.E indicates a TATA sequence followed by a CAGT or CATT mRNA start site sequence 20-40 nucleotides downstream, 180 bp upstream of the start codon.L indicates the presence of a (A/T/G) TAAG sequence.Columns 6-9 list the homologous ORF and percent amino acid identity from AcMNPV, SpltNPV-G2, SeMNPV (Spodoptera exigua multiple nucleopolyhedrovirus), and SperNPV (Spodoptera eridania nucleopolyhedrovirus), respectively

Table 2
Pairwise distances of the tandemly arranged lef8-lef9-polh nucleotide sequences were calculated by the Kimura 2-parameter model

Table 3
Identification of the ODV proteins of SpltNPV-C3.